Proteins like monoclonal antibodies (mAbs) have mostly charged and polar amino acids at the surface in an aqueous environment (Barlow, D J and Thornton, J M (1986) Biopolymers 25:1717). Because of molecular interaction with the solution components, the surface residues can undergo multiple chemical and enzymatic modifications, leading to a heterogeneous mixture of protein variants with slight differences on their electrostatic surface (Dick, L W et al., (2009) J. Chromatogr. B 877:3841; Liu, H W et al., (2008) Rapid Commun. Mass Spectrom. 22:4081; Miller, A K, et al., (2011) J. Pharm. Sci. 100:2543; Wang, W R et al., (2011) Mol. Immunol. 48:860). Cation-exchange chromatography (CEC) is considered to be the gold standard to profile the charge heterogeneity of protein therapeutics according to a recent review by Vlasak, J and Ionescu, R (2008 Curr. Pharm. Biotechnol. 9:468). The charge sensitive separation method is typically required by the regulatory agencies to ensure the production consistency during manufacturing and to monitor the degradation level of protein therapeutics (Miller, A K, et al., (2011) J. Pharm. Sci. 100:2543; He, X P Z (2009) Electrophoresis 30:714; Sosic, Z et al., (2008) Electrophoresis 29:4368; Kim, J et al., (2010) J. Chromatogr. B 878:1973: Teshima, G et al., (2010) J. Chromatogr. A 1218:2091).
Ion exchange chromatography (IEC) is typically performed in a bind and elute mode. Generally a protein sample, such as an mAb, is introduced to the stationary phase under conditions that facilitate the protein binding to the column (i.e., in 100% buffer A). A salt or pH gradient (i.e. increasing % of buffer B) is applied to induce the different charged proteins to elute in order. IEC methods are typically product specific. The development of a method that is both robust, i.e. can withstand fluctuations in temperature and pH, and can sufficiently resolve the charge heterogeneity is resource intensive. Methods to develop an optimal buffer system that allows development of robust assays to determine the presence of contaminants in multiple polypeptide products are desirable. The present invention provides methods to predict optimal conditions for ion exchange based on mathematical modeling of both the polypeptide and the buffering system.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.